circ_0006789 promotes cervical cancer development via the miR-615-5p/HSF1 axis

Objective Circular RNAs (circRNAs) are involved in the development of human cancers, including cervical cancer (CC). However, the role and mechanism of circ_0006789 (circSLC25A43) in CC are unclear. The purpose of this study was to investigate the functional role of circ_0006789 in CC. Methods The expression of circ_0006789 in CC tissues and cell lines was examined by RT-qPCR. The characterization of circ_0006789 in CC cells was verified by subcellular localisation, actinomycin D assay, and RNase R assay. After circ_0006789 was knocked down in CC cell lines, the proliferation, apoptosis, migration and invasion of CC cells were assessed by CCK-8 method, flow cytometry, and Transwell assay. RIP assay, FISH assay, dual luciferase reporter gene assay and Western blot were used to investigate the regulatory mechanism between circ_0006789, miR-615-5p and heat shock factor 1 (HSF1). Results circ_0006789 was upregulated in CC tissues and cell lines. CC cells were inhibited in their proliferation, migration, and invasion, as well as promoted to apoptosis when circ_0006789 was knocked down. It was found that circ_0006789 targeted miR-615-5p, and miR-615-5p expression was inversely correlated with circ_0006789 expression. Furthermore, HSF1 was a target gene of miR-615-5p. Furthermore, the suppressive effects on HeLa cells mediated by circ_0006789 knockdown were counter-balanced when miR-615-5p was knocked down and HSF1 was overexpressed. Mechanistically, circ_0006789 was found to promote CC development by reducing miR-615-5p and increasing HSF1 expressions. Conclusion circ_0006789 accelerates CC development via the miR-615-5p/HSF1 axis.


Introduction
Cervical cancer (CC) is a prevalent gynecological neoplasm with increasing incidence and mortality rates, particularly among younger individuals.It continues to be a highly perilous gynecological malignancy worldwide, particularly in developing nations, posing a significant threat to human well-being [1].Despite substantial progress in surgical interventions, chemotherapy, and radiotherapy, the 5-year survival rate for patients remains alarmingly low [2].The pathogenesis of CC is governed by a diverse array of molecules, and comprehending the molecular mechanisms involved is crucial.
Circular RNAs (circRNAs) possess covalent closed-loop structures [3], which do not have 5ʹ-3ʹ polarity and polyadenylate termini.Moreover, the unique circular structure of circRNAs makes them resistant to RNase and relatively stable in tissues and cells.circRNAs are aberrantly expressed in cancer cells in a tissue-specific manner, making them promising biomarkers and therapeutic targets for tumors [4][5][6][7].Research has indicated that circRNAs are implicated in the carcinogenesis, including CC, and can function as ceRNAs by modulating the activity of miRNAs [8,9].Previous investigations have identified multiple dysregulated circRNAs associated with tumorigenesis and metastasis in CC, such as circ_101996 [10], circ_0018289 [11], and circ_0000228 [12].circ_0006789 (circSLC25A43) is a circular RNA originating from the SLC25A43 gene and is known to exert a significant influence on tumor progression.Circ_0006789 was originally thought to be able to regulate the growth, migration and invasion of hepatocellular carcinoma cells by modulating the miR-1324/SOX 12 axis [13], but no studies have yet explored circ_0006789's role in CC.Various miRNAs have been implicated in CC development, either as tumor suppressors or oncogenes [14][15][16].Notably, miR-615-5p has been implicated in ovarian cancer [17].HSF1 promotes tumor progression and tumorigenesis in cancer cells by promoting their survival and escaping programmed cell death [18][19][20].Also, it has been reported that HSF1 participates in the pathogenesis of CC [21].
The objective of this study was to examine circ_0006789 expression and its biological role in CC.Our findings detected a significant upregulation of circ_0006789 in both CC tissues and cell lines.Additionally, we investigated the impact of circ_0006789 on CC development and demonstrated that circ_0006789 targets and sequesters HSF1 through miR-615-5p, thereby facilitating CC progression.

Patients and tissue samples
CC tissues (n = 150), along with adjacent normal tissues, were collected from CC patients at The First Affiliated Hospital of Harbin Medical University between January 2021 and May 2023.Histopathology biopsy confirmation was required, followed by pathology confirmation after surgery, to determine whether a patient met the inclusion criteria.None of the subjects had undergone radiation, chemotherapy, or any treatments.The specimens were stored at − 80 °C.The clinical characteristics of the patients can be found in Table 1.Furthermore, all patients provided written informed consent.This study was endorsed by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University (No. 202010HS6).

Actinomycin D experiment
By adding 2 mg/ml actinomycin D (Sigma-Aldrich), HeLa and Siha cells were incubated at 37 °C for 4, 8, 12, and 24 h.Extracted RNA was collected using HiScript II first strand cDNA synthesis kit (Vazyme, Nanjing, China) to determine the stability of circ_0006789 using PCR.

RNAse R
The extraction of total RNA (2 μg) from HeLa and Siha cells was conducted using TRIzol reagent (Invitrogen).Subsequently, digestion was carried out at 37 °C for 10 min with 3 U/μg RNAse R (Epicentre, WI, USA).Following RNA purification with RNeasy MinElute Cleanup kit (Qiagen, Hilden, Germany), PCR was conducted to assess circ_0006789 and SLC25A43 levels.

PCR
Total RNA was isolated from using TRIzol reagents (Thermo Fisher Scientific).circ_0006789 and HSF1 were treated with PrimeScript™ RT Master Mix (Takara, Shiga, Japan), while miR-615-5p was detected with miRNA reverse transcription kit (TaKaRa).PCR was performed using the SYBR® Premix Ex TaqTM II kit (TaKaRa) on the StepOnePlus realtime PCR system (Thermo Fisher Scientific).GAPDH and U6 were internal parameters to calculate the relative gene expression by 2 −ΔΔCt method.The primers are listed in Table 2.

Transwell
Transwell chambers (Corning, MA, USA) were utilized for Transwell assays.For the cell migration assay, a suspension of 1 × 10 5 HeLa and SiHa cells in serum-free medium (150 µL) was transferred to the upper compartment.Following incubation at 37 °C for 24 h, the cells on the lower compartment were fixed with 95% ethanol and stained with 0.1% crystal violet for a duration of 25 min.The cells were enumerated utilizing a microscope manufactured by Olympus, Japan.A Matrigel layer was pre-covered in the Transwell chamber to perform invasion assay.

Immunoblot
The isolation of total protein from tissues and cells was performed using RIPA lysis buffer (Beyotime, Shanghai, China), followed by quantification analysis using the BCA kit (Beyotime).Subsequently, protein samples (30 μg/lane) were separated through sodium dodecyl sulphate-polyacrylamide gel electrophoresis and subsequently transferred to a

Xenotransplantation model
Five-week-old female BALB/c nude mice (Beijing Laboratory Animal Center, Beijing, China) were utilized to construct the xenotransplantation model.HeLa cells were transfected with circ_0006789 or si-NC (RiboBio, Guangzhou.China) and selected using purinomycin.Subsequently, each mouse was given a subcutaneous inoculation of stably transfected HeLa cells (5 × 10 6 cells) into the lateral abdomen (n = 6/group).The tumor volume was assessed weekly for a duration of 4 weeks.Volume (mm 3 ) = length × width2/2.Under isoflurane anesthesia, the mice were euthanized after completing the experiment.The tumor samples were weighed, and then circ_0006789, miR-615-5p and HSF1 were detected by PCR and immunoblot.Animal treatment conformed to the approval by the Animal Management and Use Committee of The First Affiliated Hospital of Harbin Medical University (No. 202012B01).

Data analysis
All data were expressed as the mean ± standard deviation (SD) of more than 3 replicates and analyzed using GraphPad Prism 9.0 (GraphPad, CA, USA).One-way analysis of variance was employed for multiple-group differences, and Student's t-test was used for comparing two-group differences.Chi-square test was utilized to evaluate the relationship between circ_0006789 expression and clinicopathological information.* P < 0.05 was considered statistically significant.

Circ_0006789 expression and clinicopathological features
To detect circ_0006789 levels in CC, 150 paired patient's tissue samples were collected.PCR data determined that circ_0006789 expression was significantly enhanced in CC tissues (Fig. 1A).circ_0006789 levels in CC cell lines (SiHa, HeLa, HCC94, and SW756) were higher than in H8 cells (Fig. 1B).The localization of circ_0006789 in cells was further examined to determine its function in CC.Cytoplasmic separation test revealed that circ_0006789 was mainly distributed in the cytoplasm of HeLa cells (Fig. 1C).RNAse R experiments demonstrated that circ_0006789 exhibited resistance to RNAse R, whereas SLC25A43 expression was significantly diminished following RNAse R treatment (Fig. 1D).This observation suggests that circ_0006789 is a circular transcript.Furthermore, when subjected to actinomycin D treatment, circ_0006789 displayed insensitivity, further supporting its circular nature (Fig. 1E).Therefore, circ_0006789 may be involved in the corresponding molecular regulation and function post-transcriptionally.Based on circ_0006789 levels, the patients were categorized into two groups: those with high circ_0006789 expression and those with low circ_0006789 expression.circ_0006789 levels were found to be correlated with tumor differentiation and TNM stage, while no significant correlation was observed with age or lymph node metastasis (Table 2).

Knockdown of circ_0006789 significantly inhibits CC cell proliferation, migration and invasion and promotes apoptosis
HeLa cells were subjected to transfection with si-circ_0006789.Following transfection, a notable decrease in circ_0006789 expression was observed, indicating a significant efficacy of si-circ_0006789 transfection (Fig. 2A).The assessment of cell proliferation capacity was conducted using the CCK-8 method, while cell apoptosis rate was determined through flow cytometry analysis.circ_0006789 deficiency resulted in a substantial reduction in cell proliferation capacity and an enhancement in apoptosis in HeLa cells (Fig. 2B, C).Transwell assay was employed to investigate the impact of circ_0006789 on the migratory and invasive abilities of CC cells.Notably, circ_0006789 depletion resulted in suppression of migration and invasion in HeLa cells (Fig. 2D).Furthermore, immunoblot analysis was conducted to assess the

Forcing HSF1 saves the suppressive impact of circ_0006789 knockdown on CC cells
To further investigate the biological role of circ_0006789 in CC through its regulation of HSF1, we conducted functional rescue experiments.HeLa cells were transfected with either control siRNA, si-circ_0006789, or si-circ_0006789 along with a pc-HSF1 overexpression plasmid.PCR analysis revealed that transfection with si-circ_0006789 resulted in a decrease in HSF1 expression, whereas co-transfection with pc-HSF1 led to an increase in HSF1 expression (Fig. 6A).Additionally, CCK-8 demonstrated that elevating HSF1 mitigated circ_0006789 knockdown-mediated HeLa cell proliferation (Fig. 6B).Flow cytometry results determined that elevating HSF1 reduced the pro-apoptotic effect of circ_0006789 knockdown on HeLa cells (Fig. 6C).Transwell results indicated that the suppressive effect of circ_0006789 knockdown on CC cell migration and invasion was counter-balanced by overexpressing HSF1 (Fig. 6D).Immunoblot analysis results analyzed that forcing HSF1 up-regulated E-cadherin and decreased N-cadherin and Vimentin, reversing the effect of circ_0006789 knockdown on these proteins (Fig. 6E).The results demonstrated a reduction in tumor volume and weight in the si-circ_0006789 group (Fig. 7A, B).On day 28, the si-circ_0006789 group exhibited significant reductions in circ_0006789 and HSF1 levels, while miR-615-5p levels were enhanced (Fig. 7C-F).

Discussion
CircRNA is considered a biomarker relevant to clinical trials and may contribute to poor prognosis in various tumors due to its stable structure.Studies have demonstrated the significant role of circRNA as a key regulator in CC [22].For instance, circ_0000228 facilitates the malignancy of CC through the miR-195-5p/Lysyl-oxidase-like protein 2 axis [23], while circ_0000285 downregulation inhibits CC via the miR-197-3p-ELK1 axis [24].Additionally, circ_0003221 has been found to promote CC through the miR-758-3p/CPEB axis [25].The present study observed circ_0006789 up-regulation in both CC tissues and cell lines.Also, depleting circ_0006789 significantly hindered CC cells to proliferate, migrate, and invade, while concurrently enhancing apoptosis.These findings strongly imply that circ_0006789 possesses oncogenic properties and holds promise as a therapeutic target for CC treatment.However, the mechanism of action of CC treatment through circ_0006789 needs more exploration.circRNA can function as a molecular sponge, exerting indirect control over gene expression through miRNA sequestration [26].A new mechanism by which non-coding RNA regulates biological processes is suggested by the ceRNA hypothesis.The interaction between miRNA and circRNA can regulate the malignancy and aggressiveness of cancer

Fig. 1 Fig. 2
Fig. 1 circ_0006789 expression in CC.A PCR measured circ_0006789 in CC and adjacent normal tissues.B PCR measured circ_0006789 in CC cells (SiHa, HeLa, HCC94 and SW756) and normal cervical epithelial cells (H8).C Nucleocytoplasmic separation experiment detected circ_0006789 in cytoplasm and nucleus.D RNAse R assay detected the stability of circ_0006789.E PCR measured circ_0006789 after actinomycin D treatment at different time points.Data are expressed as mean ± SD (N = 3), * P < 0.05

Fig. 4
Fig. 4 Effect of circ_0006789 regulating miR-615-5p expression in CC development.A PCR detected miR-615-5p after transfection in HeLa cells.B CCK-8 assay detected the proliferation of HeLa cells.C Flow cytometry detected the apoptosis of HeLa cells.D Transwell assay detected the migration and invasion ability of HeLa cells.E Immunoblot measured Vimentin, E-cadherin, and N-cadherin in HeLa cells.Data are expressed as mean ± SD (N = 3), * P < 0.05

Fig. 5
Fig. 5 Targeting relationship between miR-615-5p and HSF1.A TargetScan predicted the binding sites of miR-615-5p and HSF1.B, C PCR detected HSF1 in CC tissues and cell lines.D Immunoblot detected HSF1 in CC tissues.E Immunoblot detected HSF1 in HeLa cells.F, G Dual luciferase reporter gene analysis and RIP analysis verified the targeting relationship between miR-615-5p and HSF1.H, I PCR and immunoblot detected HSF1 in HeLa cells.Data are expressed as mean ± SD (N = 3), * P < 0.05